RESUMO
Acoustic levitation, which allows contactless manipulation of micro-objects with ultrasounds, is a promising technique for spheroids formation and culture. This acoustofluidic technique favors cell-cell interactions, away from the walls of the chip, which leads to the spontaneous self-organization of cells. Using this approach, we generated spheroids of mesenchymal stromal cells, hepatic and endothelial cells, and showed that long-term culture of cells in acoustic levitation is feasible. We also demonstrated that this self-organization and its dynamics depended weakly on the acoustic parameters but were strongly dependent on the levitated cell type. Moreover, spheroid organization was modified by actin cytoskeleton inhibitors or calcium-mediated interaction inhibitors. Our results confirmed that acoustic levitation is a rising technique for fundamental research and biotechnological industrial application in the rapidly growing field of microphysiological systems. It allowed easily obtaining spheroids of specific and predictable shape and size, which could be cultivated over several days, without requiring hydrogels or extracellular matrix.
Assuntos
Células-Tronco Mesenquimais , Esferoides Celulares , Humanos , Células Endoteliais , Acústica , Matriz ExtracelularRESUMO
A multi-node acoustofluidic chip working on a broadband spectrum and beyond the resonance is designed for cell manipulations. A simple one-dimensional (1D) multi-layer model is used to describe the stationary standing waves generated inside a cavity. The transmissions and reflections of the acoustic wave through the different layers and interfaces lead to the creation of pressure nodes away from the resonance condition. A transparent cavity and a broadband ultrasonic transducer allow the measurement of the acoustic energy over a wide frequency range using particle image velocimetry measurements and the relation between acoustic energy and the particles velocity. The automation of the setup allows the acquisition over a large spectrum with a high frequency definition. The results show a wide continuous operating range for the acoustofluidic chip, which compares well with the 1D model. The variation of the acoustic radiation force when varying the frequency can be compensated to ensure a constant amplitude for the ARF. This approach is finally applied to mesenchymal stem cell (MCS) spheroids cultured in acoustic levitation. The MSC spheroids can be moved and merged just by varying the acoustic frequency. This approach opens the path to various acoustic manipulations and to complex 3D tissue engineering in acoustic levitation.
Assuntos
Acústica , Som , Transdutores , Ultrassom , VibraçãoRESUMO
In recent years, 3D cell culture models such as spheroid or organoid technologies have known important developments. Many studies have shown that 3D cultures exhibit better biomimetic properties compared to 2D cultures. These properties are important for in-vitro modeling systems, as well as for in-vivo cell therapies and tissue engineering approaches. A reliable use of 3D cellular models still requires standardized protocols with well-controlled and reproducible parameters. To address this challenge, a robust and scaffold-free approach is proposed, which relies on multi-trap acoustic levitation. This technology is successfully applied to Mesenchymal Stem Cells (MSCs) maintained in acoustic levitation over a 24-h period. During the culture, MSCs spontaneously self-organized from cell sheets to cell spheroids with a characteristic time of about 10 h. Each acoustofluidic chip could contain up to 30 spheroids in acoustic levitation and four chips could be ran in parallel, leading to the production of 120 spheroids per experiment. Various biological characterizations showed that the cells inside the spheroids were viable, maintained the expression of their cell surface markers and had a higher differentiation capacity compared to standard 2D culture conditions. These results open the path to long-time cell culture in acoustic levitation of cell sheets or spheroids for any type of cells.
Assuntos
Acústica , Técnicas de Cultura de Células/métodos , Diferenciação Celular , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Esferoides Celulares , Engenharia Tecidual/métodos , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , Fluoresceínas , Humanos , Propídio , Fatores de TempoRESUMO
We present a novel non-contact system for monitoring the heart rate on human subjects with clothes. Our approach is based on vibrocardiography, and measures locally skin displacements. Vibrocardiography with a laser Doppler vibrometer already allows monitoring of this vital sign, but can only be used on bare skin and requires an expensive piece of equipment. We propose here to use an airborne pulse-Doppler ultrasound system operating in the 20-60 kHz range, and comprised of an emitter focusing the ultrasound pulses on skin and a microphone recording the reflected waves. Our implementation was validated in vitro and on two healthy human subjects, using simultaneously laser vibrocardiography and electrocardiography as references. Accurate measurements of the heart rate on clothed skin suggest that our non-contact ultrasonic method could be implemented both inside and outside the clinical environment, and therefore benefit both medical and safety applications.